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Rough Sets on State Spaces of Automata
Staněk, David
This paper discussed a subclass of finite automata, which have ordering on the state sets created by a transition (or next-state) function. Hence, there do not exist cycles of more than one element. We discuss a relation of equality of upper closure on the systems of all subsets of state systems of quasi-automata, which creates an equivalence.
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Influence of transcription regulatory elemets on pre-mRNA splicing
Volek, Martin ; Staněk, David (advisor) ; Malík, Radek (referee)
In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...
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Importance of 5'-end structures of eukaryotic mRNA molecules
Vopálenský, Václav ; Pospíšek, Martin (advisor) ; Lukeš, Julius (referee) ; Staněk, David (referee)
73 V. SHRNUTÍ VÝSLEDKŮ IRESITE: THE DATABASE OF EXPERIMENTALLY VERIFIED IRES STRUCTURES (WWW.IRESITE.ORG) IRESite je kurátorovaná relační databáze typu MySQL-4.1 využívající InnoDB tabulky. Do databáze je možné vložit položky dvojího typu: o natural položky obsahují veškerá experimentální data týkající se určité IRES struktury (kompletní sekvence mRNA s vymezením pozic IRES sekvence a kódovaného proteinu, veškeré proteiny interagující s IRES, případná sekundární struktura) o engineered položky popisují uměle vytvořené plazmidy. Většinou se jedná o bicistronní plazmidy sloužící k testování funkčnosti sekvence, o níž se předpokládá, že obsahuje IRES element, a případné mutantní deriváty této sekvence. Do IRESite se v tomto případě vkládají údaje o sekvenci mRNA s vymezením IRES oblasti, informace o reportérových proteinech, informace o translačních experimentech včetně použitého promotoru a informace o pozitivních a negativních kontrolách. V databázi je nyní uloženo 288 položek; 67 natural (20 virových a 47 buněčných) a 221 engineered. IRESite slouží nejen k ukládání experimentálních dat, ale umožňuje také jejich prohledávání podle klíčových slov, případně následné porovnávání vybraných experimentů. A BIOINFORMATICAL APPROACH TO THE ANALYSIS OF VIRAL AND CELLULAR INTERNAL RIBOSOME ENTRY Publikace...
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Molecular principles of translation reinitiation in mammals
Hronová, Vladislava ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Translation initiation is a multistep process resulting in the formation of the elongation-competent 80S ribosome at the AUG start codon of the mRNA to be translated into a polypeptide chain. This process is orchestrated by numerous proteins called eukaryotic initiation factors (eIFs), out of which the most multitasking one is the eukaryotic initiation factor 3 (eIF3). The main focus of our laboratory aims at the complex characterization of the multisubunit protein eIF3 and the mechanisms of its contribution to various steps of translation initiation. Besides this, we also study one of the gene-specific translational control mechanisms called reinitiation which was, at least in yeast, also shown to be promoted by eIF3. Here I show that the N-terminal domain (NTD) of the largest subunit of yeast eIF3, a/Tif32, plays an important role not only in anchoring the eIF3 complex to the 40S small ribosomal subunit but it also critically contributes to mRNA recruitment to the 43S preinitiation complexes in vivo. The mRNA stabilization role of the a/Tif32-NTD at the mRNA exit channel of the 40S subunit was further confirmed in our following study by biophysical experiments. There, using in vivo approaches, we also demonstrated that mRNAs with longer 5'UTRs are more dependent on the stabilization role of the...
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The role of pre-mRNA splicing in human hereditary diseases
Malinová, Anna ; Staněk, David (advisor) ; Vanáčová, Štěpánka (referee) ; Krásný, Libor (referee)
U5 small ribonucleoprotein particle (U5 snRNP) is a crucial component of the spliceosome, the complex responsible for pre-mRNA splicing. Despite the importance of U5 snRNP, not much is known about its biogenesis. When we depleted one of the core U5 components, protein PRPF8, the other U5-specific proteins do not associate with U5 snRNA and the incomplete U5 was accumulated in nuclear structures known as Cajal bodies. To further clarify the role of PRPF8 in U5 snRNP assembly, we studied PRPF8 mutations that cause an autosomal dominant retinal disorder, retinitis pigmentosa (RP). We prepared eight different PRPF8 variants carrying RP-associated mutations and expressed them stably in human cell culture. We showed that most mutations interfere with the assembly of snRNPs which consequently leads to reduced efficiency of splicing. The mutant PRPF8 together with EFTUD2 are stalled in the cytoplasm in a form of U5 snRNP assembly intermediate. Strikingly, we identified several chaperons including the HSP90/R2TP complex and ZNHIT2 as new PRPF8's interactors and potential U5 snRNP assembly factors. Our results further imply that these chaperons preferentially bind the unassembled U5 complexes and that HSP90 is required for stability of...
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The role of translation initiation factor 3 (eIF3) in translation termination.
Beznosková, Petra ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
Protein synthesis is a tightly regulated process of gene expression. Each gene has its start and its stop, which is determined by one of the three stop codons. Many recent articles describe ribosomes that purposely bypass stops on specific mRNAs to extend the nascent polypeptide to alter its properties. It is called programmed stop codon readthrough. Since over 15% of human genetic diseases are caused by so called premature termination codons (PTC) that halt translation and produce truncated proteins, this mechanism has a great potential implication in medical research. Numerous labs search for non-toxic drugs specifically increasing readthrough at PTCs; however, the success of this effort requires identification and understanding of all factors that are involved in this process. Here, we present one such factor eukaryotic initiation factor 3 (eIF3) and describe its ability to induce readthrough on stop codons in termination non-favorable context during programmed readthrough and also the consequences of its action on translation regulation. We additionally analyzed which near-cognate (nc) tRNAs are incorporated at UGA stop codons depending on the nucleotide that immediately follows them (so called +4 base). This way we established new rules for stop codon decoding and identified so called...
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Role of promoter in the regulation of alternative splicing
Kozáková, Eva ; Staněk, David (advisor) ; Půta, František (referee) ; Blažek, Dalibor (referee)
It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...
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Formation of splicing machinery in the context of the cell nucleus
Stejskalová, Eva ; Staněk, David (advisor) ; Vanáčová, Štěpánka (referee) ; Malínský, Jan (referee)
Most of the protein coding genes of higher eukaryotes contain introns which have to be removed from primary transcripts to make mRNA which can be used as a template for protein synthesis. This crucial step in the pre-mRNA processing is carried out by the spliceosome, a complex ribonucleoprotein machine formed from small ribonucleoprotein particles (snRNPs). snRNPs biogenesis is a complex process composed of several steps which take place in both the cytoplasm and the nucleus. Spliceosome assembly is highly dynamic and tightly regulated and pre-mRNA splicing depends not only on the sequence of the pre-mRNA itself but also on the nuclear context, such as the chromatin modifications. How do cells regulate where and when the spliceosome would be assembled? What determines which introns will be spliced? These are fundamental, yet unanswered, biological questions. In this work we analyzed the formation of splicing machinery in the context of the cell nucleus from several different points of view. First, we investigated the unexpected connection between splicing factor U1-70K and the survival of motor neurons (SMN) complex which is a major player in the snRNP biogenesis pathway. We revealed that U1-70K interacts with the SMN complex and that this interaction is crucial for the stability of nuclear gems, small...
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Functional and biochemical characterization of elF3 and elF3j in Saccharomyces cerevisiae and human cell lines
Wagner, Susan ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Staněk, David (referee)
3 Abstract 3.1 english Translation is divided into initiation, elongation, termination and ribosome recycling. One of the largest eukaryotic initiation factors (eIF), eIF3, plays a role in nearly all steps of initiation and was recently also implicated in ribosomal recycling. Here we uncovered novel roles for eIF3 in translation termination in yeast, where the five core eIF3 subunits and their loosely associated eIF3j/HCR1 accessory factor control stop codon read-through in the opposite manner. In addition, we further characterized the function of eIF3j in initiation. Structural analysis revealed that the conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). This binding mode was shown to be conserved in yeast ensuring efficient 40S-binding by eIF3 and stringency of AUG recognition, where j/HCR1 seems to co-operate with eIF1A. We found that the N-terminal half of j/HCR1 in yeast is sufficient for fulfilling all functions of the full-length protein necessary for wild-type growth. Despite the logical dispensability of the j/HCR1 C- terminal half, it was shown that it bears a specific KERR motif that is evolutionary conserved and contained also within the HCR1-like domain of a/TIF32, through which it...
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